No Depletion Due to Incorrect Probe Design Input

Symptom

Percentage of reads mapping to the targeted sequence does not decrease after depletion treatment. Sequencing analysis shows target RNA remains at original levels.

1. 1 cDNA sequence was used as input in NEBNext Custom RNA Depletion Design Tool instead of RNA sequence
2. 2 Probes do not cover the genomic region being evaluated for depletion assessment
3. 3 Gaps (>15 nt) exist between probe coverage and high-read-coverage areas in the target
4. 4 Probe orientation is incorrect - probes must be reverse complement to target sequences

1. 1 Verify sequence source is RNA (not cDNA) before inputting into the design tool
2. 2 Align probes against target sequence using Bowtie or bwa, visualize with IGV or IGB to identify coverage gaps
3. 3 Design additional probes against gap regions using Custom RNA Depletion Tool and spike into original pool
4. 4 Run no-treatment control for comparison when testing modified probe pools
5. 5 Note: small gaps (~15 nt) between probes are expected and acceptable as fragments will be eliminated during library cleanup

Related Video (2)

RocheSequencingUSA ★ 72

The Importance of Performing Ribosomal RNA Depletion

"Demonstrates proper rRNA depletion methodology and expected outcomes, providing context for recognizing when depletion fails"

the bumbling biochemist ★ 68

rRNA depletion strategies

"Covers rRNA depletion strategies and methods, helping researchers understand correct technical approaches to avoid input format errors"